Interdisciplinary Graduate Programs Symposium
2014 OSU Molecular Life Sciences
Interdisciplinary Graduate Programs Symposium
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Talk abstracts
Abstract not available online - please check the printed booklet.
Abstract:
Photolyase is a photoenzyme which utilizes blue light to repair UV-damaged DNA. The catalytic cofactor of photolyase, flavin adenine dinucleotide, has an unusual bent configuration in photolyase, and such a folded structure may have a functional role in initial photochemistry. Using femtosecond spectroscopy, we report here our systematic characterization of cyclic intramolecular electron transfer (ET) dynamics between the flavin and adenine moieties of flavin adenine dinucleotide in four redox forms of the oxidized, neutral, and anionic semiquinone, and anionic hydroquinone states. By comparing wild-type and mutant enzymes, we have determined that the excited neutral oxidized and semiquinone states absorb an electron from the adenine moiety in 19 and 135 ps, whereas the excited anionic semiquinone and hydroquinone states donate an electron to the adenine moiety in 12 ps and 2 ns, respectively. All back ET dynamics occur ultrafast within 100 ps. These four ET dynamics dictate that only the anionic hydroquinone flavin can be the functional state in photolyase due to the slower ET dynamics (2 ns) with the adenine moiety and a faster ET dynamics (250 ps) with the substrate, whereas the intervening adenine moiety mediates electron tunneling for repair of damaged DNA. Assuming ET as the universal mechanism for photolyase and cryptochrome, these results imply anionic flavin as the more attractive form of the cofactor in the active state in cryptochrome to induce charge relocation to cause an electrostatic variation in the active site and then lead to a local conformation change to initiate signaling.
Keywords: DNA repair, electron transfer, photolyase
Abstract not available online - please check the printed booklet.
Abstract:
Geminiviruses replicate ssDNA genomes through dsDNA intermediates that associate with cellular histones to form mini-chromosomes. We previously showed viral mini-chromosomes can be targeted for repressive cytosine methylation and can acquire repressive histone methylation (e.g. di-methylated lysine 9 on Histone H3, H3K9me2). Conversely, viral genomes lacking cytosine methylation are often associated with active histone marks (e.g. Histone H3 acetylated at lysine 9/14, H3Ac). While components involved in histone post-translational modification in plants are relatively well characterized, less is known about the proteins that read these epigenetic marks. Histone reader proteins bind specific histone modifications to maintain and/or spread a modification, and can alter gene expression by recruiting specific nucleosome remodeling complexes and transcription factors. Using geminiviruses as models to identify and characterize plant chromatin readers, we hypothesize EMSY LIKE (EML) proteins 1 & 3 are plant histone readers. EML1 and EML3 contain Agenet domains homologous to known histone reader domains (Tudor domains). Initial studies tested whether these proteins interact with histones and geminivirus genomes. To date, eml1 and eml3 Arabidopsis plants display either hypersusceptibility or increased tolerance to Cabbage leaf curl virus (CaLCuV) compared to wild-type plants. Differences in symptoms correlate with changes in viral DNA levels, as measured by qPCR analysis. Using chromatin immunoprecipitation (ChIP) we have also observed changes in H3K9me2 and H3K9Ac levels associated with CaLCuV genomes in mutant plants. Co-immunoprecipitation and ChIP experiments further indicate EML1 and EML3 associate with histone H3 and the CaLCuV genome. These results suggest Agenet domain-containing EML proteins are histone readers and play specific roles in promoting virus infection or bolstering plant antiviral defense pathways.
References:
1. Raja P, Sanville BC, Buchmann RC & DM Bisaro. (2008). Viral genome methylation as an epigenetic defense against geminiviruses. J Virol. 82(18):8997-9007.
2. Mauer-Stroh S, Dickens NJ, Hughes-Davies L, Kouzarides T, Eisenhaber F & CP Ponting. (2003). The Tudor domain ‘Royal Family’: Tudor, plant Agenet, Chromo, PWWP and MBT domains. Trends Biochem Sci. 28(2):69-74.
Keywords: Chromatin, Geminiviruses, Molecular Virolgoy
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Dr. Ann Altman received her undergraduate degree in the Natural Sciences from the University of Cambridge (UK) and her doctorate, in Molecular Biophysics and Biochemistry, from Yale University. After a dozen years as a researcher at the Yale University School of Medicine, she hit the "glass ceiling" and left academia to start her own editorial business. She has edited more than 8000 scientific manuscripts and written four books for young scientists, in particular, "Guide to Publishing a Scientific Paper," which forms the basis of her lecture (a pdf is attached). If you decide to Google her, you will find her scientific contributions under her maiden name, Ann M. Körner.
Keywords: scientific writing
Abstract:
Our study aims to understand mitotic targeting of proteins to the nuclear envelope (NE). During mitosis in the model fungus Aspergillus nidulans, peripheral nuclear pore complex (NPC) proteins (Nups) disperse from NPCs thereby opening nuclear pores allowing nuclear entry of mitotic regulators. Paradoxically, one highly conserved peripheral Nup involved in nuclear export, Gle1, does not disperse from the NE during mitosis. Hence, we investigated how Gle1 is tethered to the mitotic NE. For this, we performed Gle1 affinity purifications and found that it co-purifies specifically with AN0162, which has a C-terminal transmembrane domain, suggesting AN0162 might tether Gle1 to the mitotic NE membrane. Deletion experiments revealed that Gle1 associates with NPCs during interphase but leaves them during mitosis to associate with AN0162 at the inner nuclear membrane (INM).
To identify structural features within AN0162 required for its INM targeting and Gle1 interaction, we replaced endogenous AN0162 with red fluorescent protein-tagged truncated versions in a strain expressing endogenous Gle1-GFP. This enabled tracking of the AN0162 truncations and their effect on Gle1 targeting during the cell cycle. This approach identified several domains within AN0162 required for its INM targeting including the C-terminal transmembrane domain and a domain sufficient for nuclear localization. A Gle1 binding domain (GBD) was also identified. Versions of AN0162 lacking its GBD were targeted to the INM normally but failed to target Gle1 to the INM during mitosis. Conversely, strains lacking INM targeting domains of AN0162, but retaining the GBD, modified mitotic Gle1 distribution. Our data therefore reveal the existence of distinct and alternating cell cycle regulated mechanisms for NE protein targeting. Interestingly cells lacking AN0162 have rounder nuclei, while cells overexpressing AN0162 have larger and highly misshapen nuclei. Thus AN0162 functions to target Gle1 to the INM only during mitosis and is important to maintain normal overall nuclear structure.
References:
1. De Souza, C.P., et al., Partial nuclear pore complex disassembly during closed mitosis in Aspergillus nidulans. Curr Biol, 2004. 14(22): p. 1973-84.
2. De Souza, C.P. and S.A. Osmani, Double duty for nuclear proteins--the price of more open forms of mitosis. Trends Genet, 2009. 25(12): p. 545-54.
3. De Souza, C.P. and S.A. Osmani, Mitosis, not just open or closed. Eukaryot Cell, 2007. 6(9): p. 1521-7.
Keywords: mitosis, protein targeting, nuclear envelope
Abstract:
Amyotrophic Lateral Sclerosis (ALS) is an adult onset neurodegenerative disease, characterized by progressive and fatal loss of motor neurons. Mutations in the Superoxide dismutase 1 (SOD1) gene are one of the leading causes of familial ALS while misfolded wild-type SOD1 is suggested to be involved in the pathogenesis of sporadic ALS. Here, we determined the feasibility and efficacy of post-natal downregulation of SOD1 as a therapeutic strategy in ALS, using a novel approach of Adeno Associated Virus Serotype 9 (AAV9)-mediated shRNA delivery. SOD1G93A mice, overexpressing human mutant SOD1, were injected intravenously at post-natal day 1 (P1), P21 or P85 with AAV9-SOD1 shRNA. P1-treated mice showed persistent transduction of AAV9-SOD1 shRNA in motor neurons and astrocytes while P21 and P85 treated mice showed predominant astrocytic transduction. Injected mice showed reduced levels of mutant SOD1 in the spinal cord. Both P1 and P21 injected mice showed improved performance on behavioral tasks. P1 injected mice showed significant delay in the disease onset and progression while P21 and P85 injected mice had a significant delay in disease progression. Importantly, all treatments significantly extended the median survival of SOD1G93A mice by 30-50 days. Injection of AAV9-SOD1 shRNA, even after the disease onset, in slow progressing SOD1G37R mice resulted in 84 days extension in the median survival. Further, AAV9-SOD1 shRNA administration in wild-type mice revealed no adverse effects. Finally, intrathecal administration of AAV9-SOD1 shRNA in non-human primates (Cynomolgus monkeys) resulted in efficient spinal cord transduction and significant SOD1 reduction. Thus, the success of post-natal suppression of SOD1 toxicity in ALS mice, long-term safety assessment in wild-type mice and robust SOD1 knockdown in non-human primates sets the stage for future clinical trials. Moreover, the involvement of SOD1 in familial and sporadic ALS underlines the potential of this approach as a gene therapy for ALS.
References:
Boillee, S., Vande Velde, C. & Cleveland, D.W. ALS: a disease of motor neurons and their nonneuronal neighbors. Neuron 52, 39-59 (2006).
Boillee, S., et al. Onset and progression in inherited ALS determined by motor neurons and microglia. Science 312, 1389-1392 (2006).
Yamanaka, K., et al. Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis. Nat Neurosci 11, 251-253 (2008).
Bosco, DA., et al. Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS. Nat Neurosci 13: 1396–1403 (2010).
Haidet-Phillips, AM., et al. Astrocytes from familial and sporadic ALS patients are toxic to motor neurons. Nat Biotechnol 29, 824–828 (2011).
Foust, K.D., et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol 27, 59-65 (2009).
Keywords: Amyotrophic Lateral Sclerosis (ALS), Adeno Associated Virus Serotype 9 (AAV9), Superoxide Dismutase 1 (SOD1)
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Retroviruses must integrate their reverse transcribed DNA into host chromatin in order to replicate. However, sites of integration are not random and vary by retroviral genera. For example; lentiviruses, such as human immunodeficiency virus type I (HIV-1), target integration to actively transcribed genes, whereas γ-retroviruses, such as murine leukemia virus (MLV), target transcription start sites. Preferential site selection of retroviral integration is known to be mediated through interactions between retroviral integrase and different host transcription factors. My research seeks to understand the structural mechanisms behind this tethering. Lens epithelium-derived growth factor/p75 (LEDGF/p75) is the human protein that HIV-1 hijacks for integration. The binding of LEDGF/p75 to HIV-1 integrase occurs through a C-terminal domain termed the integrase binding domain (IBD), while binding of LEDGF/p75 to host chromatin occurs though the PWWP domain named for its conserved Pro-Trp-Trp-Pro motif. We determined the NMR solution structure of the LEDGF/p75 PWWP domain and have characterized the binding of this domain to chromatin by performing NMR titrations for binding to a peptide containing tri-methylated K36 from histone H3 (H3K36me3) or double-stranded DNA. The resulting chemical shift perturbations identified the sites of peptide and DNA binding. We have also determined the binding affinity for each by fluorescence anisotropy. Binding of the PWWP domain to H3K36me3 and DNA explains the site selection of HIV-1, since the H3K36me3 is an epigenetic mark for actively transcribed genes. In contrast to the findings for lentiviral integration, MLV hijacks a different transcription factor family, the bromodomain and extraterminal domain (BET) family. The BET family contains two N-terminal bromodomains, which bind and recognize acetylated lysines of histone tails, which are marks for transcription start sites, and an extraterminal (ET) domain which has recently been found to bind to MLV integrase directly. We used NMR chemical shift perturbations to determine the binding site on Brd4 ET domain of a peptide from MLV integrase. These studies have provided unique insights into the structural mechanisms retroviruses utilize to target integration to specific sites within human chromatin.
References:
Eidahl, J. O., Crowe, B. L., North, J. A., McKee, C. J., Shkriabai, N., Feng, L., Plumb, M., Graham, R. L., Gorelick, R. J., Hess, S., Poirier, M. G., Foster, M. P., and Kvaratskhelia, M. (2013) Nucleic acids research 41, 3924-3936
Larue, R. C., Plumb, M. R., Crowe, B. L., Shkriabai, N., Sharma, A., Difiore, J., Malani, N., Aiyer, S. S., Roth, M. J., Bushman, F. D., Foster, M. P., and Kvaratskhelia, M. (2014) Nucleic acids research
Keywords: Retrovirus integration, Epigenetics, NMR solution structure
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract not available online - please check the printed booklet.
Abstract:
Deregulated expression of microRNAs has been shown to occur in acute myeloid leukemia (AML) and contribute to the disease aggressiveness. In AML low expression of miR-181a associated with worse outcomes but the exact mechanisms of how miR-181a is mediating this effect remains elusive. Aberrant activation of the RAS-pathway, by mutation or overexpression is also frequent in AML. We hypothesized that miR-181a’s anti-leukemic activity might in part mediate by downregulating the RAS-pathway. Here we showed that the role of miR-181a in modulating the aggressiveness of disease, in vitro and in vivo. Increased miR-181a levels prolonged survival in a murine leukemia model. We showed that miR-181a targets NRAS, KRAS and MAPK1 involved in the oncogenic RAS signaling cascade that support aberrant malignant cell proliferation. To exploit these findings therapeutically we formulated targeted nanoparticles to deliver synthetic miR-181a to AML cells. miR-181a-nanoparticle treatment increased mature miR-181a on average >140-fold and >35-fold in AML cell lines and primary patient blasts, respectively. Delivered miR-181a downregulated KRAS, NRAS and MAPK1 and in turn resulted in decreased phosphorylation of down-stream RAS effectors i.e., AKT and MEK; this in turn results in downregulation of the MAKP1-target oncogenic transcription factor MYC. miR-181a-nanoparticle treatment led to an anti-leukemia activity. It reduced proliferation, impaired colony formation and sensitized AML cells to the chemotherapeutic daunorubicin. Finally, in an AML mouse model miR-181a-nanoparticle treatment resulted in a significantly longer survival. In conclusion, anti-leukemic miR-181a targets the RAS/MAPK-signaling pathway. Treatment with miR-181a-nanoparticles increased miR-181a levels and provides anti-leuemia activity. This may represent a novel therapeutic approach for AML patients.
Keywords: miR-181a, Nanoparticles, Acute Myeloid Leukemia
Abstract:
Interest in the function of numerous post-transcriptional modifications of tRNA bases and sugars that are known to occur in all three domains of life has increased in recent years. While translation-related roles for some modified nucleotides found near the tRNA anticodon are relatively well-established, the biological function of many modifications found in the remaining tRNA body is far less well-understood. Some modifications occurring in the core of the tRNA affect overall stability, and thus, loss of specific modifications may lead to degradation. In addition, cells exposed to oxidative stress or growth arrest may gain additional modifications on certain tRNAs. These data suggest that tRNA modification can be regulated in cells as a way to ensure overall quality and function of the tRNA pool, but the consequences of alternative tRNA modification patterns remain to be fully investigated.
In this work, we show that the yeast m1G9 methyltransferase, Trm10, displays the ability to modify additional tRNA substrates, both in vitro and in vivo, beyond the set of tRNA species that are normally modified in wild-type S. cerevisiae. We hypothesize that this expanded mode of substrate specificity is advantageous in that it could allow Trm10 to modify non-cognate tRNAs in cells under stress, possibly preserving the structural integrity of the tRNA. Using in vitro activity assays with tRNA chimera, we have identified tRNA elements that affect substrate specificity of yeast Trm10. Moreover, analysis of the modification status of the 5'-end of tRNAs exposed to two different stress conditions using primer extension revealed additional primer extension stops corresponding to positions of known tRNA modifications, including N-1 methylation at G9. These data support the hypothesis that the modification status of tRNAs is much more dynamic than previously understood and opens the door to further investigation of the physiological function of alternative tRNA modification patterns in cells.
Keywords: tRNA modification, tRNA structure, specificity
Abstract:
Accounting for around 6% of all cases of Duchenne muscular dystrophy, exon duplications provide an excellent avenue for new exon skipping therapies. We sought to test the efficacy of virally-mediated duplication skipping in the novel Dup2 mouse, modeling the most common single-exon duplication (exon 2) seen in DMD patients. A targeting construct was created containing four copies of a modified U7snRNA, each of which targets either the splice donor or acceptor sites of exon 2 (U7sn RNA-ACCA). Both tibilias anterior IM injections and tail vein IV injections were done in 8 week mice and then analyzed 4 weeks later at both the mRNA and protein level. RT-PCR reveals widespread exon 2 skipping, with the simultaneous presence of all 3 predicted transcripts – duplicated exon 2, wild-type, and deleted exon 2 – in variable proportions. Dystrophin expression and location was confirmed by immunoblot as well as by immunofluorescence. Treatment normalized hindlimb and forelimb grip strength and partially corrected extensor digitorum longus force deficits seen in untreated Dup2 mice. These results demonstrate the utility of the Dup2 mouse model as a tool for testing potential duplication exon-skipping strategies. They confirm that IV delivery of rAAV9.U7snRNA is able induce exon 2 skipping and to drive the production of a functional dystrophin protein, suggesting a promising strategy for future clinical development.
Keywords: AAV, muscle, therapy
Abstract:
The cell cycle is frequently dysregulated in breast cancer, leading to uncontrolled division of cells. It is tightly controlled by cyclins, cyclin-dependent kinases (CDKs), and their downstream target, retinoblastoma protein (Rb). Dramatic clinical data seen recently with the small-molecule CDK4/6 inhibitor palbociclib (PB-0332991) targeting estrogen receptor (ER)-positive breast cancer, which accounts for 75% of all breast cancers, has rekindled interest in the concept of blocking cell cycle progression to stop cancer cell growth. In this regard, we tested the efficacy of the multikinase CDK inhibitor, PHA-848125 in breast cancer. Cell proliferation assays after PHA-848125 treatment of a large panel of breast cancer cells showed a reduction in the growth rate exclusively in ER-negative breast cancer cell lines, but not ER-positive cell lines. As a result of the CDK2 inhibitory activity of the drug, cell cycle analysis revealed a specific G1 arrest with a concomitant reduction of the phosphorylation of Rb. Inhibitory activity of the drug was also observed in vivo by treatment of ER-negative MDA-MB-231 xenotransplanted tumors with 40mg/kg of PHA-848125 twice a day, 5 days a week, for three weeks. Interestingly, repeated cycles of treatment did not induce resistance to the drug, which is the main reason of failure of many chemotherapies. Finally, treatment with PHA-848125 for 10 days of MMTV-PyVT transgenic mice that developed multifocal mammary adenocarcinoma and lung metastatic lesions showed tumor growth inhibition and a strong reduction in metastasic colonization of lungs. The results were further confirmed by orthotopic transplantation of MDA-MB-231 in NOD-SCID mice. Overall, these experiments demonstrated that PHA-848125 specifically target ER-negative breast cancer both in vitro and in vivo. Our report of the first targeted therapy for the ER-negative subtype lays out the rationale for the clinical evaluation of PHA-848125 in therapy for breast cancer.
Keywords: CDK inhibitor, PHA-848125, breast cancer
